CRISPR/Cas: aplicaciones y perspectivas para el mejoramiento genético de plantas

The rapid development of CRISPR/Cas technology in recent years, from its discovery as a microbial immune system to its evolution as a versatile and powerful tool for the modification of gene function, constitutes a statement on how basic research with a moderate impact can revolutionize biological investigations. From its application as a platform for RNA-guided gene editing, the CRISPR/Cas system has spread to fields as diverse as the regulation of gene expression, epigenome editing, and in vivo visualization of chromatin. In this article the CRISPR/Cas technology is introduced, with emphasis on the CRISPR/Cas9 type II system and its main applications in plant breeding are discussed. Likewise, advances and limitations in the use of this technology in plants are highlighted, and future perspectives in the field of genetic improvement are outlined.Keywords:abiotic and biotic stress, gene function modification, genetic engineering, genomeEl desarrollo vertiginoso de la tecnología CRISPR/Cas en los últimos años, desde su descubrimiento como sistema inmune microbiano hasta su evolución como herramienta versátil y potente para la modificación de la función génica, constituye un manifiesto sobre cómo investigaciones básicas de impacto moderado pueden revolucionar las investigaciones biológicas. A partir de su aplicación como plataforma para la edición génica guiada por ARN, el sistema CRISPR/Cas se ha extendido hacia campos tan diversos como la regulación de la expresión génica, edición del epigenoma y visualización in vivo de la cromatina. En este artículo se introduce la tecnología CRISPR/Cas con énfasis en el sistema de tipo II CRISPR/Cas9 y se discuten susprincipales aplicaciones en el mejoramiento genético en plantas. Igualmente, se destacan los avances y limitaciones del uso de la tecnología en plantas, y se delinean las futuras perspectivas en el campo del mejoramiento genético. Palabras clave:edición de genoma, estrés biótico y abiótico, ingeniería genética, modificación de función génic

Perfil de expresión de una lipasa de tipo GDSL durante interacciones compatible e incompatible entre Musa spp. y Pseudocercospora fijiensis

The identification of genes of interest for the genetic improvement of Musa spp. against Pseudocercospora fijiensis Morelet is constrained by the limited knowledge of the molecular mechanisms of the plant-pathogen interaction. GDSL-like lipases are promising targets since they are related to the mechanisms of resistance and susceptibility to pathogens. The aim of this work was to determine, through quantitative PCR, the expression profiles of a GDSL-like lipase gene from Musa acuminata Colla during compatible and incompatible interactions with P. fijiensis. Based on this analysis, the gene coding for the GDSL-like lipase was identified in the banana genome and its overexpression was verified in the compatible interaction at the early stage of infection.Keywords:black Sigatoka, Mycosphaerella fijiensis, quantitative PCRLa identificación de genes de interés para el mejoramiento genético de Musa spp. contra Pseudocercospora fijiensis Morelet se ve limitada por el escaso conocimiento de los mecanismos moleculares de la interacción planta-patógeno. Las lipasas de tipo GDSL constituyen blancos promisorios al estar relacionadas con los mecanismos de resistencia y suceptibilidad a patógenos. El objetivo de este trabajo fue determinar mediante PCR cuantitativo los perfiles de expresión del gen de una lipasa de tipo GDSL de Musa acuminata Colla durante las interacciones compatible e incompatible con P. fijiensis. A partir del análisis realizado se identificó el gen que codifica para la lipasa de tipo GDSL en el genoma de banano y se comprobó su sobreexpresión en la interacción compatible en estadio temprano de infección.Palabras clave:Mycosphaerella fijiensis, PCR cuantitativo, Sigatoka negr

Genome-Wide classification and phylogenetic analyses of the GDSL-Type Esterase/Lipase (GELP) family in flowering plants

GDSL-type esterase/lipase (GELP) enzymes have key functions in plants, such as developmental processes, anther and pollen development, and responses to biotic and abiotic stresses. Genes that encode GELP belong to a complex and large gene family, ranging from tens to more than hundreds of members per plant species. To facilitate functional transfer between them, we conducted a genome-wide classification of GELP in 46 plant species. First, we applied an iterative phylogenetic method using a selected set of representative angiosperm genomes (three monocots and five dicots) and identified 10 main clusters, subdivided into 44 orthogroups (OGs). An expert curation for gene structures, orthogroup composition, and functional annotation was made based on a literature review. Then, using the HMM profiles as seeds, we expanded the classification to 46 plant species. Our results revealed the variable evolutionary dynamics between OGs in which some expanded, mostly through tandem duplications, while others were maintained as single copies. Among these, dicot-specific clusters and specific amplifications in monocots and wheat were characterized. This approach, by combining manual curation and automatic identification, was effective in characterizing a large gene family, allowing the establishment of a classification framework for gene function transfer and a better understanding of the evolutionary history of GELP

Expression profile of a GDSL-like lipase during compatible and incompatible interactions between <i>Musa</i> spp. and <i>Pseudocercospora fijiensis</i>

The identification of genes of interest for the genetic improvement of Musa spp. against Pseudocercospora fijiensis Morelet is constrained by the limited knowledge of the molecular mechanisms of the plant-pathogen interaction. GDSL-like lipases are promising targets since they are related to the mechanisms of resistance and susceptibility to pathogens. The aim of this work was to determine, through quantitative PCR, the expression profiles of a GDSL-like lipase gene from Musa acuminata Colla during compatible and incompatible interactions with P. fijiensis. Based on this analysis, the gene coding for the GDSL-like lipase was identified in the banana genome and its overexpression was verified in the compatible interaction at the early stage of infection. Keywords:black Sigatoka, Mycosphaerella fijiensis, quantitative PC

Effect of inoculation time on <i>Agrobacterium</i>-mediated transformation efficiency of <i>Musa</i> cv. ‘Grande naine’ (AAA)

Increasing inoculation time during Agrobacterium-mediated transformation has been shown to favour transformation efficiency in several plant species. The effect of inoculation time in combination with spermidine (Spd) on efficiency was determined during Agrobaterium-mediated transformation of the Musa cultivar ‘Grande naine’ (AAA). Banana embryogenic cell suspensions (ECSs) were incubated with the bacterial strain EHA 105 carrying the binary vector pFAJ3000. Six different inoculation conditions (6 h, 6 h+Spd, 12 h, 12 h+Spd, 24 h, 24 h+Spd) were compared based on transient GUS expression and number of embryo colonies formed. Moreover, leaf fragments from 24 randomly chosen regenerated plantlets were assayed presence and expression of the transgenes. Consequently, samples that were inoculated for 24 h in medium supplemented with 1 mM spermidine showed the highest transformation efficiency, expressed as number of blue foci and regenerated colonies after the selection treatment. Here we showed for the first time that longer inoculation times in combination with spermidine enhance the efficiency of Agrobacterium-mediated transformation of the banana cultivar ‘Grande naine’.    Keywords: banana, β-glucuronidase, genotype, somatic embryo, PC